Natural killer cells and cytotoxic T-lymphocytes are important in the immune response against virally infected cells and tumor cells. Cell mediated death occurs by two distinct mechanisms, fas/fas-ligand and granule exocytosis. One of the major components of the cytotoxic granules are serine-proteases, termed granzymes. Although the role of granzymes in causing cell death has not been established, recent experiments wit granzymeB-deficient mice show it is critical for target DNA fragmentation. We have cloned rat granzyme B, C, and two other granzymes that have not yet been classified. Three of these granzymes have been expressed in Pichia pastoris. The subsite specificity of Granzyme B has been determined and corresponds to the activation site of the caspase family of cysteine proteases that are implicated in cell death by apoptosis. Apart from the biological significance, this series of homologous serine proteases presents a system for elucidating the structural determinants of substrate specificity. Additionally, a serine collagenase isolated from fiddler crab is being used to probe the interaction with its substrate, collagen. The Computer Graphics Laboratory is used to structurally model these proteases which provides insight into the structural determinants of substrate specificity. These models are used to guide the redesign of the granzymes.